Premenopausal women are relatively protected against clinically apparent coronary heart disease (CHD), compared to age-matched males. Although this difference has been accepted to be hormonally-mediated, this concept has been challenged recently by results of prospective, large clinical trials and cohort studies. Many questions have now been raised about the therapeutic/prophylactic approach to CHD in postmenopausal women. This has intensified the interest in a finer dissection of both the molecular and physiologic aspects of estrogen's effects on the vasculature. CHD is the result of a dynamic process that includes modulation of vasoreactivity, vascular injury, cell growth and matrix deposition, processes affected by endothelium-derived nitric oxide (NO). In addition to its effects on gene expression, estrogen, through engagement of membrane receptors, stimulates rapid signaling. Our hypothesis is that there exist membrane-initiated effects of estrogen on the endothelium, resulting in rapid endothelial activation, promoting a relatively non-atherogenic environment. We have recently demonstrated that 17 beta-estradiol (E2) activates endothelial NO synthase in a c-Src/phosphatidylinositol-3 kinase/Akt-dependent fashion, and that this is most efficiently mediated by an endothelial cell (EC) membrane-localized ERalpha isoform, ER46. Specific proposals now include: (1) to dissect the proximal components of the E2-stimulated, membrane ER (mER)-mediated signaling cascade in human EC by addressing phosphatase-mediated activation of c-Src, heterotrimeric G protein involvement, requirement for mER dimerization, and performing a proteomics screen for E2-induced ER46-associated proteins; (2) to define mER localization, topology and structure by mutating critical ER46 domains (palmitoylation, transmembrane), and to purify sufficient quantities of mER46 to eventually enable crystallization, using a yeast expression system; (3) to evaluate the role of c-Src in E2-stimulated, endothelium/NO-dependent responses in/ex vivo, using murine models of ear vascular permeability, cremaster muscle arteriolar vasomotion and aortic ring tension in c-Src -/- mice, using the ERalpha-Neo, betaKO mouse (expressing only ER46) to demonstrate functionality of this receptor in vivo; and (4) to test the efficiency hierarchy of selective estrogen receptor modulators (SERMs), with regard to c-Src/Akt activation and NO release, in a complete ER knockout mouse EC line reconstituted with human ER46. These studies will not only enhance our understanding of ER biology, but also provide more detailed molecular and physiologic information that may shed considerable light on the controversies in hormone replacement therapy. [unreadable] [unreadable]